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Covid-19, caused by the SARS-CoV-2 virus, as yet does not have a proven vaccine or drug treatment to prevent infection.  Researchers globally are studying the virus and its method of infection to develop effective vaccines and drug treatments.  Listed below are a few of the papers recently published regarding SARS-CoV-2, Covid-19, and potential therapies and treatments.

 

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COVID-19: Melatonin as a Potential Adjuvant Treatment

Rui Zhang, Xuebin Wang, Leng Ni, Xiao Di, Baitao Ma, Shuai Niu, Changwei Liu, Russel J Reiter, Life Sci2020 Jun 1;250:117583.

doi: 10.1016/j.lfs.2020.117583. Epub 2020 Mar 23.

Abstract

This article summarizes the likely benefits of melatonin in the attenuation of COVID-19 based on its putative pathogenesis. The recent outbreak of COVID-19 has become a pandemic with tens of thousands of infected patients. Based on clinical features, pathology, the pathogenesis of acute respiratory disorder induced by either highly homogenous coronaviruses or other pathogens, the evidence suggests that excessive inflammation, oxidation, and an exaggerated immune response very likely contribute to COVID-19 pathology. This leads to a cytokine storm and subsequent progression to acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and often death. Melatonin, a well-known anti-inflammatory and anti-oxidative molecule, is protective against ALI/ARDS caused by viral and other pathogens. Melatonin is effective in critical care patients by reducing vessel permeability, anxiety, sedation use, and improving sleeping quality, which might also be beneficial for better clinical outcomes for COVID-19 patients. Notably, melatonin has a high safety profile. There is significant data showing that melatonin limits virus-related diseases and would also likely be beneficial in COVID-19 patients. Additional experiments and clinical studies are required to confirm this speculation.

Design of Multi-Epitope Vaccine Candidate Against SARS-CoV-2: A in-silico Study

K Abraham Peele, T Srihansa, S Krupanidhi, A Vijaya Sai, T C Venkateswarulu, J Biomol Struct Dyn2020 Jun 1;1-9.

doi: 10.1080/07391102.2020.1770127. Online ahead of print.

Abstract

The best therapeutic strategy to find an effective vaccine against SARS-CoV-2 is to explore the target structural protein. In the present study, a novel multi-epitope vaccine is designed using in silico tools that potentially trigger both CD4 and CD8 T-cell immune responses against the novel Coronavirus. The vaccine candidate was designed using B and T-cell epitopes that can act as an immunogen and elicits immune response in the host system. NCBI was used for the retrieval of surface spike glycoprotein, of novel corona virus (SARS-CoV-2) strains. VaxiJen server screens the most important immunogen of all the proteins and IEDB server gives the prediction and analysis of B and T cell epitopes. Final vaccine construct was designed in silico composed of 425 amino acids including the 50S ribosomal protein adjuvant and the construct was computationally validated in terms of antigenicity, allergenicity and stability on considering all critical parameters into consideration. The results subjected to the modeling and docking studies of vaccine were validated. Molecular docking study revealed the protein-protein binding interactions between the vaccine construct and TLR-3 immune receptor. The MD simulations confirmed stability of the binding pose. The immune simulation results showed significant response for immune cells. The findings of the study confirmed that the final vaccine construct of chimeric peptide could able to enhance the immune response against nCoV-19.

Identification of 22 N-glycosites on Spike Glycoprotein of SARS-CoV-2 and Accessible Surface Glycopeptide Motifs: Implications for Vaccination and Antibody Therapeutics

Dapeng Zhou, Xiaoxu Tian, Ruibing Qi, Chao Peng, Wen Zhang, Glycobiology2020 Jun 10; cwaa052.

doi: 10.1093/glycob/cwaa052. Online ahead of print.

Abstract

Coronaviruses hijack human enzymes to assemble the sugar coat on their spike glycoproteins. The mechanisms by which human antibodies may recognize the antigenic viral peptide epitopes hidden by the sugar coat are unknown. Glycosylation by insect cells differs from the native form produced in human cells, but insect cell-derived influenza vaccines have been approved by the US Food and Drug Administration. In this study, we analyzed recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein secreted from BTI-Tn-5B1-4 insect cells, by trypsin and chymotrypsin digestion followed by mass spectrometry analysis. We acquired tandem mass spectrometry (MS/MS) spectrums for glycopeptides of all 22 predicted N-glycosylated sites. We further analyzed the surface accessibility of spike proteins according to cryogenic electron microscopy and homolog-modeled structures, and available antibodies that bind to SARS-CoV-1. All 22 N-glycosylated sites of SARS-CoV-2 are modified by high-mannose N-glycans. MS/MS fragmentation clearly established the glycopeptide identities. Electron densities of glycans cover most of the spike receptor-binding domain of SARS-CoV-2, except YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ, similar to a region FSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQ in SARS-CoV-1. Other surface-exposed domains include those located on central helix, connecting region, heptad repeats, and N-terminal domain. Because the majority of antibody paratopes bind to the peptide portion with or without sugar modification, we propose a snake-catching model for predicted paratopes: a minimal length of peptide is first clamped by a paratope, and sugar modifications close to the peptide either strengthen or do not hinder the binding.

Targeting the Dimerization of the Main Protease of Coronaviruses: A Potential Broad-Spectrum Therapeutic Strategy

Bhupesh Goyal, Deepti Goyal, ACS Comb Sci. 2020 Jun 8; 22(6):297-305.

doi: 10.1021/acscombsci.0c00058.Epub 2020 May 27.

Abstract

A new coronavirus (CoV) caused a pandemic named COVID-19, which has become a global health care emergency in the present time. The virus is referred to as SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) and has a genome similar (∼82%) to that of the previously known SARS-CoV (SARS coronavirus). An attractive therapeutic target for CoVs is the main protease (Mpro) or 3-chymotrypsin-like cysteine protease (3CLpro), as this enzyme plays a key role in polyprotein processing and is active in a dimeric form. Further, Mpro is highly conserved among various CoVs, and a mutation in Mpro is often lethal to the virus. Thus, drugs targeting the Mpro enzyme significantly reduce the risk of mutation-mediated drug resistance and display broad-spectrum antiviral activity. The combinatorial design of peptide-based inhibitors targeting the dimerization of SARS-CoV Mpro represents a potential therapeutic strategy. In this regard, we have compiled the literature reports highlighting the effect of mutations and N-terminal deletion of residues of SARS-CoV Mpro on its dimerization and, thus, catalytic activity. We believe that the present review will stimulate research in this less explored yet quite significant area. The effect of the COVID-19 epidemic and the possibility of future CoV outbreaks strongly emphasize the urgent need for the design and development of potent antiviral agents against CoV infections.

Virtual Screening and Repurposing of FDA Approved Drugs Against COVID-19 Main Protease

Mahmoud Kandeel, Mohammed Al-Nazawi,  Life Sci. 2020 Jun 15; 251:117627.

doi: 10.1016/j.lfs.2020.117627. Epub 2020 Apr 3.

Abstract

Aims: In December 2019, the Coronavirus disease-2019 (COVID-19) virus has emerged in Wuhan, China. In this research, the first resolved COVID-19 crystal structure (main protease) was targeted in a virtual screening study by of FDA approved drugs dataset. In addition, a knowledge gap in relations of COVID-19 with the previously known fatal Coronaviruses (CoVs) epidemics, SARS and MERS CoVs, was covered by investigation of sequence statistics and phylogenetics.

Materials and methods: Molecular modeling, virtual screening, docking, sequence comparison statistics and phylogenetics of the COVID-19 main protease were investigated.

Key findings: COVID-19 Mpro formed a phylogenetic group with SARS CoV that was distant from MERS CoV. The identity% was 96.061 and 51.61 for COVID-19/SARS and COVID-19/MERS CoV sequence comparisons, respectively. The top 20 drugs in the virtual screening studies comprised a broad-spectrum antiviral (ribavirin), anti-hepatitis B virus (telbivudine), two vitamins (vitamin B12 and nicotinamide) and other miscellaneous systemically acting drugs. Of special interest, ribavirin had been used in treating cases of SARS CoV.

Significance: The present study provided a comprehensive targeting of the first resolved COVID+19 structure of Mpro and found a suitable save drugs for repurposing against the viral Mpro. Ribavirin, telbivudine, vitamin B12 and nicotinamide can be combined and used for COVID treatment. This initiative relocates already marketed and approved safe drugs for potential use in COVID-treatment.

Chemistry and Biology of SARS-CoV-2

Alexander Dömling, Li Gao,  Chem. 2020 Jun 11; 6(6):1283-1295.

doi: 10.1016/j.chempr.2020.04.023.Epub 2020 May 22.

Abstract

SARS-CoV-2 (previously 2019-nCoV or Wuhan coronavirus) caused an unprecedented fast-spreading worldwide pandemic. Although currently with a rather low mortality rate, the virus spread rapidly over the world using the modern world’s traffic highways. The coronavirus (CoV) family members were responsible for several deadly outbreaks and epidemics during the last decade. Not only governments but also the scientific community reacted promptly to the outbreak, and information is shared quickly. For example, the genetic fingerprint was shared, and the 3D structure of key proteins was rapidly solved, which can be used for the discovery of potential treatments. An overview is given on the current knowledge of the spread, disease course, and molecular biology of SARS-CoV-2. We discuss potential treatment developments in the context of recent outbreaks, drug repurposing, and development timelines.

 

SARS-CoV-2 Genomic Surveillance in Taiwan Revealed Novel ORF8-deletion Mutant and Clade Possibly Associated With Infections in Middle East

Yu-Nong Gong , Kuo-Chien Tsao , Mei-Jen Hsiao , Chung-Guei Huang , Peng-Nien Huang, Po-Wei Huang, Kuo-Ming Lee, Yi-Chun Liu, Shu-Li Yang , Rei-Lin Kuo , Kuan-Fu Chen , Yen-Chin Liu , Sheng-Yu Huang, Hsing-I Huang , Ming-Tsan Liu , Ji-Rong Yang , Cheng-Hsun Chiu , Cheng-Ta Yang , Guang-Wu Chen , Shin-Ru Shih,  Emerg Microbes Infect2020 Jun 16; 1-37.

doi: 10.1080/22221751.2020.1782271.Online ahead of print.

Abstract

Taiwan experienced two waves of imported infections with Coronavirus Disease 2019 (COVID-19). This study aimed at investigating the genomic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Taiwan and compared their evolutionary trajectories with the global strains. We performed culture and full-genome sequencing of SARS-CoV-2 strains followed by phylogenetic analysis. A 382-nucleotides deletion in open reading frame 8 (ORF8) was found in a Taiwanese strain isolated from a patient on February 4, 2020 who had a travel history to Wuhan. Patients in the first wave also included several sporadic, local transmission cases. Genomes of 5 strains sequenced from clustered infections were classified into a new clade with ORF1ab-V378I mutation, in addition to 3 dominant clades ORF8-L84S, ORF3a-G251V and S-D614G. This highlighted clade also included some strains isolated from patients who had a travel history to Turkey and Iran. The second wave mostly resulted from patients who had a travel history to Europe and Americas. All Taiwanese viruses were classified into various clades. Genomic surveillance of SARS-CoV-2 in Taiwan revealed a new ORF8-deletion mutant and a virus clade that may be associated with infections in the Middle East, which contributed to a better understanding of the global SARS-CoV-2 transmission dynamics.

Inhibitors of SARS-CoV-2 Entry: Current and Future Opportunities

Siyu Xiu, Alexej Dick, Han Ju, Sako Mirzaie, Fatemeh Abdi, Simon Cocklin, Peng Zhan, Xinyong Liu, J Med Chem2020 Jun 15.

doi: 10.1021/acs.jmedchem.0c00502. Online ahead of print.

Abstract

Recently, a novel coronavirus initially designated 2019-nCoV but now termed SARS-CoV-2 has emerged and raised global concerns due to its virulence. SARS-CoV-2 is the etiological agent of “coronavirus disease 2019”, abbreviated to COVID-19, which despite only being identified at the very end of 2019, has now been classified as a pandemic by the World Health Organization (WHO). At this time, no specific prophylactic or post-exposure therapy for COVID-19 is currently available. Viral entry is the first step in the SARS-CoV-2 lifecycle and is mediated by the trimeric spike protein. Being the first stage in infection, entry of SARS-CoV-2 into host cells is an extremely attractive therapeutic intervention point. Within this review, we highlight therapeutic intervention strategies for anti-SARS-CoV, MERS-CoV, and other coronaviruses and speculate upon future directions for SARS-CoV-2 entry inhibitor designs.

Human Leukocyte Antigen Susceptibility Map for Severe Acute Respiratory Syndrome Coronavirus 2

Austin Nguyen, Julianne K David , Sean K Maden , Mary A Wood , Benjamin R Weeder, Abhinav Nellore , Reid F Thompson, J Virol2020 Jun 16; 94(13):e00510-20.

doi: 10.1128/JVI.00510-20. Print 2020 Jun 16.

Abstract

Genetic variability across the three major histocompatibility complex (MHC) class I genes (human leukocyte antigen A [HLA-A], -B, and -C genes) may affect susceptibility to and severity of the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19). We performed a comprehensive in silico analysis of viral peptide-MHC class I binding affinity across 145 HLA-A, -B, and -C genotypes for all SARS-CoV-2 peptides. We further explored the potential for cross-protective immunity conferred by prior exposure to four common human coronaviruses. The SARS-CoV-2 proteome was successfully sampled and was represented by a diversity of HLA alleles. However, we found that HLA-B*46:01 had the fewest predicted binding peptides for SARS-CoV-2, suggesting that individuals with this allele may be particularly vulnerable to COVID-19, as they were previously shown to be for SARS (M. Lin, H.-T. Tseng, J. A. Trejaut, H.-L. Lee, et al., BMC Med Genet 4:9, 2003, https://bmcmedgenet.biomedcentral.com/articles/10.1186/1471-2350-4-9). Conversely, we found that HLA-B*15:03 showed the greatest capacity to present highly conserved SARS-CoV-2 peptides that are shared among common human coronaviruses, suggesting that it could enable cross-protective T-cell-based immunity. Finally, we reported global distributions of HLA types with potential epidemiological ramifications in the setting of the current pandemic. IMPORTANCE Individual genetic variation may help to explain different immune responses to a virus across a population. In particular, understanding how variation in HLA may affect the course of COVID-19 could help identify individuals at higher risk from the disease. HLA typing can be fast and inexpensive. Pairing HLA typing with COVID-19 testing where feasible could improve assessment of severity of viral disease in the population. Following the development of a vaccine against SARS-CoV-2, the virus that causes COVID-19, individuals with high-risk HLA types could be prioritized for vaccination.

SARS-CoV-2 nsp13, nsp14, nsp15 and orf6 Function as Potent Interferon Antagonists

Chun-Kit Yuen, Joy-Yan Lam , Wan-Man Wong Long-Fung Mak, Xiaohui Wang, Hin Chu , Jian-Piao Cai, Dong-Yan Jin , Kelvin Kai-Wang To, Jasper Fuk-Woo Chan , Kwok-Yung Yuen, Kin-Hang Kok, Emerg Microbes Infect2020 Jun 12; 1-29.

doi: 10.1080/22221751.2020.1780953.Online ahead of print.

Abstract

The Coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2 virus, is now causing a tremendous global health concern. Since its first appearance in December 2019, the outbreak has already caused over 5.8 million infections worldwide (till 29 May 2020), with more than 0.35 million deaths. Early virus-mediated immune suppression is believed to be one of the unique characteristics of SARS-CoV-2 infection and contributes at least partially to the viral pathogenesis. In this study, we identified the key viral interferon antagonists of SARS-CoV-2 and compared them with two well-characterized SARS-CoV interferon antagonists, PLpro and orf6. Here we demonstrated that the SARS-CoV-2 nsp13, nsp14, nsp15 and orf6, but not the unique orf8, could potently suppress primary interferon production and interferon signaling. Although SARS-CoV PLpro has been well-characterized for its potent interferon-antagonizing, deubiquitinase and protease activities, SARS-CoV-2 PLpro, despite sharing high amino acid sequence similarity with SARS-CoV, loses both interferon-antagonising and deubiquitinase activities. Among the 27 viral proteins, SARS-CoV-2 orf6 demonstrated the strongest suppression on both primary interferon production and interferon signaling. Orf6-deleted SARS-CoV-2 may be considered for the development of intranasal live-but-attenuated vaccine against COVID-19.

Boceprevir, GC-376, and Calpain Inhibitors II, XII Inhibit SARS-CoV-2 Viral Replication by Targeting the Viral Main Protease

Chunlong Ma, Michael Dominic Sacco, Brett Hurst, Julia Alma Townsend , Yanmei Hu, Tommy Szeto, Xiujun Zhang, Bart Tarbet, Michael Thomas Marty, Yu Chen, Jun Wang, Cell Res2020 Jun 15.

doi: 10.1038/s41422-020-0356-z. Online ahead of print.

Abstract

A new coronavirus SARS-CoV-2, also called novel coronavirus 2019 (2019-nCoV), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.35% as of May 26, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (Mpro). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC50 values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC50 values ranging from 0.49 to 3.37 µM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known substrate-based peptidomimetic Mpro inhibitors. A complex crystal structure of SARS-CoV-2 Mpro with GC-376, determined at 2.15 Å resolution with three protomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by Mpro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.

Molecular Simulation of SARS-CoV-2 Spike Protein Binding to Pangolin ACE2 or Human ACE2 Natural Variants Reveals Altered Susceptibility to Infection

Jingfang Wang, Xintian Xu, Xinbo Zhou, Ping Chen, Huiying Liang, Xuan Li, Wu Zhong, Pei Hao, J Gen Virol2020 Jun 15.

doi: 10.1099/jgv.0.001452. Online ahead of print.

Abstract

We constructed complex models of SARS-CoV-2 spike protein binding to pangolin or human ACE2, the receptor for virus transmission, and estimated the binding free energy changes using molecular dynamics simulation. SARS-CoV-2 can bind to both pangolin and human ACE2, but has a significantly lower binding affinity for pangolin ACE2 due to the increased binding free energy (9.5 kcal mol-1). Human ACE2 is among the most polymorphous genes, for which we identified 317 missense single-nucleotide variations (SNVs) from the dbSNP database. Three SNVs, E329G (rs143936283), M82I (rs267606406) and K26R (rs4646116), had a significant reduction in binding free energy, which indicated higher binding affinity than wild-type ACE2 and greater susceptibility to SARS-CoV-2 infection for people with them. Three other SNVs, D355N (rs961360700), E37K (rs146676783) and I21T (rs1244687367), had a significant increase in binding free energy, which indicated lower binding affinity and reduced susceptibility to SARS-CoV-2 infection.

Potential Antiviral Options Against SARS-CoV-2 Infection

Aleksandr Ianevski, Rouan Yao, Mona Høysæter Fenstad, Svetlana Biza, Eva Zusinaite, Tuuli Reisberg, Hilde Lysvand, Kirsti Løseth, Veslemøy Malm Landsem, Janne Fossum Malmring, Valentyn Oksenych, Sten Even Erlandsen, Per Arne Aas, Lars Hagen, Caroline H Pettersen, Tanel Tenson, Jan Egil Afset, Svein Arne Nordbø, Magnar Bjørås, Denis E Kainov, Viruses2020 Jun 13; 12(6):E642.

doi: 10.3390/v12060642.

Abstract

As of June 2020, the number of people infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) continues to skyrocket, with more than 6.7 million cases worldwide. Both the World Health Organization (WHO) and United Nations (UN) has highlighted the need for better control of SARS-CoV-2 infections. However, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against SARS-CoV-2 can be time-consuming and costly. Convalescent sera and safe-in-man broad-spectrum antivirals (BSAAs) are readily available treatment options. Here, we developed a neutralization assay using SARS-CoV-2 strain and Vero-E6 cells. We identified the most potent sera from recovered patients for the treatment of SARS-CoV-2-infected patients. We also screened 136 safe-in-man broad-spectrum antivirals against the SARS-CoV-2 infection in Vero-E6 cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. We found that a combination of orally available virus-directed nelfinavir and host-directed amodiaquine exhibited the highest synergy. Finally, we developed a website to disseminate the knowledge on available and emerging treatments of COVID-19.

Withanone and Withaferin-A Are Predicted to Interact With Transmembrane Protease Serine 2 (TMPRSS2) and Block Entry of SARS-CoV-2 Into Cells

Vipul Kumar, Jaspreet Kaur Dhanjal, Priyanshu Bhargava, Ashish Kaul, Jia Wang, Huayue Zhang, Sunil C Kaul, Renu Wadhwa, Durai Sundar, J Biomol Struct Dyn2020 Jun 16; 1-13.

doi: 10.1080/07391102.2020.1775704. Online ahead of print.

Abstract

Coronavirus disease 2019 (COVID-19) initiated in December 2019 in Wuhan, China and became pandemic causing high fatality and disrupted normal life calling world almost to a halt. Causative agent is a novel coronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2/2019-nCoV). While new line of drug/vaccine development has been initiated world-wide, in the current scenario of high infected numbers, severity of the disease and high morbidity, repurposing of the existing drugs is heavily explored. Here, we used a homology-based structural model of transmembrane protease serine 2 (TMPRSS2), a cell surface receptor, required for entry of virus to the target host cell. Using the strengths of molecular docking and molecular dynamics simulations, we examined the binding potential of Withaferin-A (Wi-A), Withanone (Wi-N) and caffeic acid phenethyl ester to TPMRSS2 in comparison to its known inhibitor, Camostat mesylate. We found that both Wi-A and Wi-N could bind and stably interact at the catalytic site of TMPRSS2. Wi-N showed stronger interactions with TMPRSS2 catalytic residues than Wi-A and was also able to induce changes in its allosteric site. Furthermore, we investigated the effect of Wi-N on TMPRSS2 expression in MCF7 cells and found remarkable downregulation of TMPRSS2 mRNA in treated cells predicting dual action of Wi-N to block SARS-CoV-2 entry into the host cells. Since the natural compounds are easily available/affordable, they may even offer a timely therapeutic/preventive value for the management of SARS-CoV-2 pandemic. We also report that Wi-A/Wi-N content varies in different parts of Ashwagandha and warrants careful attention for their use.Communicated by Ramaswamy H. Sarma.

Computational Determination of Potential Inhibitors of SARS-CoV-2 Main Protease

Son Tung Ngo, Ngoc Quynh Anh Pham, Ly Thi Le, Duc-Hung Pham, Van V Vu, J Chem Inf Model2020 Jun 12.

doi: 10.1021/acs.jcim.0c00491. Online ahead of print.

Abstract

The novel coronavirus (SARS-CoV-2) has infected several million people and caused thousands of deaths worldwide since Dec 2019. As the disease is spreading rapidly all over the world, it is urgent to find effective drugs to treat the virus. The main protease (Mpro) of SARS-CoV-2 is one of the potential drug targets. Therefore, in this context, we used rigorous computational methods, including molecular docking, fast pulling of ligand (FPL), and free energy perturbation (FEP), to investigate potential inhibitors of SARS-CoV-2 Mpro. We first tested our approach with three reported inhibitors of SARS-CoV-2 Mpro; and our computational results are in good agreement with the respective experimental data. Subsequently, we applied our approach on a databases of ~4600 natural compounds, as well as 8 available HIV-1 protease (PR) inhibitors and an aza-peptide epoxide. Molecular docking resulted in a short list of 35 natural compounds, which was subsequently refined using the FPL scheme. FPL simulations resulted in five potential inhibitors, including 3 natural compounds and two available HIV-1 PR inhibitors. Finally, FEP, the most accurate and precise method, was used to determine the absolute binding free energy of these five compounds. FEP results indicate that two natural compounds, cannabisin A and isoacteoside, and an HIV-1 PR inhibitor, darunavir, exhibit large binding free energy to SARS-CoV-2 Mpro, which is larger than that of 13b, the most reliable SARS-CoV-2 Mpro inhibitor recently reported. The binding free energy largely arises from van der Waals interaction. We also found that Glu166 form H-bonds to all the inhibitors. Replacing Glu166 by an alanine residue leads to ~ 2.0 kcal/mol decreases in the affinity of darunavir to SARS-CoV-2 Mpro. Our results could contribute to the development of potentials drugs inhibiting SARS-CoV-2.

Eltrombopag Is a Potential Target for Drug Intervention in SARS-CoV-2 Spike Protein

Siqin Feng, Xiaodong Luan, Yifei Wang, Hui Wang, Zhiyu Zhang, Yiyang Wang, Zhuang Tian, Meixi Liu, Ying Xiao, Yong Zhao, Ruilin Zhou, Shuyang Zhang, Infect Genet Evol2020 Jun 12;104419.

doi: 10.1016/j.meegid.2020.104419. Online ahead of print.

Abstract

The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is a current global threat for which there is an urgent need to search for an effective therapy. The transmembrane spike (S) glycoprotein of SARS-CoV-2 directly binds to the host angiotensin-converting enzyme 2 (ACE2) and mediates viral entrance, which is therefore considered as a promising drug target. Considering that new drug development is a time-consuming process, drug repositioning may facilitate rapid drug discovery dealing with sudden infectious diseases. Here, we compared the differences between the virtual structural proteins of SARS-CoV-2 and SARS-CoV, and selected a pocket mainly localizing in the fusion cores of S2 domain for drug screening. A virtual drug design algorithm screened the Food and Drug Administration-approved drug library of 1234 compounds, and 13 top scored compounds were obtained through manual screening. Through in vitro molecular interaction experiments, eltrombopag was further verified to possess a high binding affinity to S protein plus human ACE2 and could potentially affect the stability of the ACE2-S protein complex. Hence, it is worth further exploring eltrombopag as a potential drug for the treatment of SARS-CoV-2 infection.

Structural Analysis of the Putative SARS-CoV-2 Primase Complex

Eva Konkolova, Martin Klima, Radim Nencka, Evzen Boura, J Struct Biol2020 Jun 11; 211(2):107548.

doi: 10.1016/j.jsb.2020.107548. Online ahead of print.

Abstract

We report the crystal structure of the SARS-CoV-2 putative primase composed of the nsp7 and nsp8 proteins. We observed a dimer of dimers (2:2 nsp7-nsp8) in the crystallographic asymmetric unit. The structure revealed a fold with a helical core of the heterotetramer formed by both nsp7 and nsp8 that is flanked with two symmetry-related nsp8 β-sheet subdomains. It was also revealed that two hydrophobic interfaces one of approx. 1340 Å2 connects the nsp7 to nsp8 and a second one of approx. 950 Å2 connects the dimers and form the observed heterotetramer. Interestingly, analysis of the surface electrostatic potential revealed a putative RNA binding site that is formed only within the heterotetramer.

De novo Design of Protein Peptides to Block Association of the SARS-CoV-2 Spike Protein With Human ACE2

Xiaoqiang Huang, Robin Pearce Yang Zhang, Aging (Albany NY)2020 Jun 16;12.

doi: 10.18632/aging.103416. Online ahead of print.

Abstract

The outbreak of COVID-19 has now become a global pandemic that has severely impacted lives and economic stability. There is, however, no effective antiviral drug that can be used to treat COVID-19 to date. Built on the fact that SARS-CoV-2 initiates its entry into human cells by the receptor binding domain (RBD) of its spike protein binding to the angiotensin-converting enzyme 2 (hACE2), we extended a recently developed approach, EvoDesign, to design multiple peptide sequences that can competitively bind to the SARS-CoV-2 RBD to inhibit the virus from entering human cells. The protocol starts with the construction of a hybrid peptidic scaffold by linking two fragments grafted from the interface of the hACE2 protein (a.a. 22-44 and 351-357) with a linker glycine, which is followed by the redesign and refinement simulations of the peptide sequence to optimize its binding affinity to the interface of the SARS-CoV-2 RBD. The binding experiment analyses showed that the designed peptides exhibited a significantly stronger binding potency to hACE2 than the wild-type hACE2 receptor (with -53.35 vs. -46.46 EvoEF2 energy unit scores for the top designed and wild-type peptides, respectively). This study demonstrates a new avenue to utilize computationally designed peptide motifs to treat the COVID-19 disease by blocking the critical spike-RBD and hACE2 interactions.

Identification of Potential Natural Inhibitors of SARS-CoV2 Main Protease by Molecular Docking and Simulation Studies

Sanjay Gupta, Atul Kumar Singh, Prem Prakash Kushwaha, Kumari Sunita Prajapati, Mohd Shuaib, Sabyasachi Senapati, Shashank Kumar, J Biomol Struct Dyn. 2020 Jun 11;1-12.

doi: 10.1080/07391102.2020.1776157. Online ahead of print.

Abstract

Coronaviruses are contagious pathogens primarily responsible for respiratory and intestinal infections. Research efforts to develop antiviral agents against coronavirus demonstrated the main protease (Mpro) protein may represent effective drug target. X-ray crystallographic structure of the SARS-CoV2 Mpro protein demonstrated the significance of Glu166, Cys141, and His41 residues involved in protein dimerization and its catalytic function. We performed in silico screening of compounds from Curcuma longa L. (Zingiberaceae family) against Mpro protein inhibition. Employing a combination of molecular docking, scoring functions, and molecular dynamics simulations, 267 compounds were screened by docking on Mpro crystallographic structure. Docking score and interaction profile analysis exhibited strong binding on the Mpro catalytic domain with compounds C1 (1E,6E)-1,2,6,7-tetrahydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione) and C2 (4Z,6E)-1,5-dihydroxy-1,7-bis(4-hydroxyphenyl)hepta-4,6-dien-3-one as lead agents. Compound C1 and C2 showed minimum binding score (-9.08 and -8.07 kcal/mole) against Mpro protein in comparison to shikonin and lopinavir (≈ -5.4 kcal/mole) a standard Mpro inhibitor. Furthermore, principal component analysis, free energy landscape and protein-ligand energy calculation studies revealed that these two compounds strongly bind to the catalytic core of the Mpro protein with higher efficacy than lopinavir, a standard antiretroviral of the protease inhibitor class. Taken together, this structure based optimization has provided lead on two natural Mpro inhibitors for further testing and development as therapeutics against human coronavirus.

Current Perspective of Antiviral Strategies Against COVID-19

Bintou A Ahidjo, Marcus Wing Choy Loe, Yan Ling Ng, Chee Keng Mok, Justin Jang Hann Chu, ACS Infect Dis2020 Jun 16.

doi: 10.1021/acsinfecdis.0c00236. Online ahead of print.

Abstract

COVID-19 was declared a pandemic by the World Health Organization on March 11, 2020. This novel coronavirus disease, caused by the SARS-CoV-2 virus, has resulted in severe and unprecedented social and economic disruptions globally. Since the discovery of COVID-19 in December 2019, numerous antivirals have been tested for efficacy against SARS-CoV-2 in vitro and also clinically to treat this disease. This review article discusses the main antiviral strategies currently employed and summarizes reported in vitro and in vivo efficacies of key antiviral compounds in use.

Virtual Screening, ADME/Tox Predictions and the Drug Repurposing Concept for Future Use of Old Drugs Against the COVID-19

Lorane Izabel da Silva Hage-Melim , Leonardo Bruno Federico, Nayana Keyla Seabra de Oliveira, Viviane Cristina Cardoso Francisco, Lenir Cabral Correa, Henrique Barros de Lima, Suzane Quintana Gomes, Mariana Pegrucci Barcelos, Isaque Antônio Galindo Francischini, Carlos Henrique Tomich de Paula da Silva, Life Sci. 2020 Jun 11; 256:117963.

doi: 10.1016/j.lfs.2020.117963. Online ahead of print.

Abstract

The new Coronavirus (SARS-CoV-2) is the cause of a serious infection in the respiratory tract called COVID-19. Structures of the main protease of SARS-CoV-2 (Mpro), responsible for the replication of the virus, have been solved and quickly made available, thus allowing the design of compounds that could interact with this protease and thus to prevent the progression of the disease by avoiding the viral peptide to be cleaved, so that smaller viral proteins can be released into the host’s plasma. These structural data are extremely important for in silico design and development of compounds as well, being possible to quick and effectively identify potential inhibitors addressed to such enzyme’s structure. Therefore, in order to identify potential inhibitors for Mpro, we used virtual screening approaches based with the structure of the enzyme and two compounds libraries, targeted to SARS-CoV-2, containing compounds with predicted activity against Mpro. In this way, we selected, through docking studies, the 100 top-ranked compounds, which followed for subsequent studies of pharmacokinetic and toxicity predictions. After all the simulations and predictions here performed, we obtained 10 top-ranked compounds that were again in silico analyzed inside the Mpro catalytic site, together some drugs that are being currently investigated for treatment of COVID-19. After proposing and analyzing the interaction modes of these compounds, we submitted one molecule then selected as template to a 2D similarity study in a database containing drugs approved by FDA and we have found and indicated Apixaban as a potential drug for future treatment of COVID-19.

Fragment Molecular Orbital Based Interaction Analyses on COVID-19 Main Protease – Inhibitor N3 Complex (PDB ID:6LU7)

Ryo Hatada, Koji Okuwaki, Yuji Mochizuki, Yuma Handa, Kaori Fukuzawa, Yuto Komeiji, Yoshio Okiyama, Shigenori Tanaka, J Chem Inf Model2020 Jun 15.

doi: 10.1021/acs.jcim.0c00283. Online ahead of print.

Abstract

The worldwide spread of COVID-19 (new coronavirus found in 2019) is an emergent issue to be tackled. In fact, a great amount of works in various fields have been made in rather short period. Here, we report a fragment molecular orbital (FMO) based interaction analysis on a complex between the SARS-CoV-2 main protease (Mpro) and its peptide-like inhibitor N3 (PDB ID: 6LU7). The target inhibitor molecule was segmented into five fragments in order to capture site specific interactions with amino acid residues of the protease. The interaction energies were decomposed into several contributions, and then the characteristics of hydrogen bonding and dispersion stabilization were made clear. Furthermore, the hydration effect was incorporated by the Poisson-Boltzmann (PB) scheme. From the present FMO study, His41, His163, His164, and Glu166 were found to be the most important amino acid residues of Mpro in interacting with the inhibitor, mainly due to hydrogen bonding. A guideline for optimizations of the inhibitor molecule was suggested as well based on the FMO analysis.

Bioinformatic Prediction of Potential T Cell Epitopes for SARS-Cov-2

Kazuma Kiyotani, Yujiro Toyoshima, Kensaku Nemoto, Yusuke Nakamura, J Hum Genet. 2020 Jul; 65(7):569-575.

doi: 10.1038/s10038-020-0771-5. Epub 2020 May 6.

Abstract

To control and prevent the current COVID-19 pandemic, the development of novel vaccines is an emergent issue. In addition, we need to develop tools that can measure/monitor T-cell and B-cell responses to know how our immune system is responding to this deleterious virus. However, little information is currently available about the immune target epitopes of novel coronavirus (SARS-CoV-2) to induce host immune responses. Through a comprehensive bioinformatic screening of potential epitopes derived from the SARS-CoV-2 sequences for HLAs commonly present in the Japanese population, we identified 2013 and 1399 possible peptide epitopes that are likely to have the high affinity (<0.5%- and 2%-rank, respectively) to HLA class I and II molecules, respectively, that may induce CD8+ and CD4+ T-cell responses. These epitopes distributed across the structural (spike, envelope, membrane, and nucleocapsid proteins) and the nonstructural proteins (proteins corresponding to six open reading frames); however, we found several regions where high-affinity epitopes were significantly enriched. By comparing the sequences of these predicted T cell epitopes to the other coronaviruses, we identified 781 HLA-class I and 418 HLA-class II epitopes that have high homologies to SARS-CoV. To further select commonly-available epitopes that would be applicable to larger populations, we calculated population coverages based on the allele frequencies of HLA molecules, and found 2 HLA-class I epitopes covering 83.8% of the Japanese population. The findings in the current study provide us valuable information to design widely-available vaccine epitopes against SARS-CoV-2 and also provide the useful information for monitoring T-cell responses.

Renin-angiotensin System Inhibitors Improve the Clinical Outcomes of COVID-19 Patients With Hypertension

Juan Meng, Guohui Xiao, Juanjuan Zhang, Xing He, Min Ou, Jing Bi, Rongqing Yang, Wencheng Di, Zhaoqin Wang, Zigang Li, Hong Gao, Lei Liu, Guoliang Zhang, Emerg Microbes Infect2020 Dec; 9(1):757-760.

doi: 10.1080/22221751.2020.1746200.

Abstract

The dysfunction of the renin-angiotensin system (RAS) has been observed in coronavirus infection disease (COVID-19) patients, but whether RAS inhibitors, such as angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II type 1 receptor blockers (ARBs), are associated with clinical outcomes remains unknown. COVID-19 patients with hypertension were enrolled to evaluate the effect of RAS inhibitors. We observed that patients receiving ACEI or ARB therapy had a lower rate of severe diseases and a trend toward a lower level of IL-6 in peripheral blood. In addition, ACEI or ARB therapy increased CD3 and CD8 T cell counts in peripheral blood and decreased the peak viral load compared to other antihypertensive drugs. This evidence supports the benefit of using ACEIs or ARBs to potentially contribute to the improvement of clinical outcomes of COVID-19 patients with hypertension.

Design of a Peptide-Based Subunit Vaccine Against Novel Coronavirus SARS-CoV-2

Parismita Kalita, Aditya K Padhi, Kam Y J Zhang, Timir Tripathi, Microb Pathog2020 Aug;145:104236.

doi: 10.1016/j.micpath.2020.104236. Epub 2020 May 4.

Abstract

Coronavirus disease 2019 (COVID-19) is an emerging infectious disease that was first reported in Wuhan, China, and has subsequently spread worldwide. In the absence of any antiviral or immunomodulatory therapies, the disease is spreading at an alarming rate. A possibility of a resurgence of COVID-19 in places where lockdowns have already worked is also developing. Thus, for controlling COVID-19, vaccines may be a better option than drugs. An mRNA-based anti-COVID-19 candidate vaccine has entered a phase 1 clinical trial. However, its efficacy and potency have to be evaluated and validated. Since vaccines have high failure rates, as an alternative, we are presenting a new, designed multi-peptide subunit-based epitope vaccine against COVID-19. The recombinant vaccine construct comprises an adjuvant, cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes joined by linkers. The computational data suggest that the vaccine is non-toxic, non-allergenic, thermostable, with the capability to elicit a humoral and cell-mediated immune response. The stabilization of the vaccine construct is validated with molecular dynamics simulation studies. This unique vaccine is made up of 33 highly antigenic epitopes from three proteins that have a prominent role in host-receptor recognition, viral entry, and pathogenicity. We advocate this vaccine must be synthesized and tested urgently as a public health priority.

nCOV-19 Peptides Mass Fingerprinting Identification, Binding, and Blocking of Inhibitors Flavonoids and Anthraquinone of Moringa oleifera and Hydroxychloroquine

Muhammad Hamza, Ashaq Ali , Suliman Khan, Saeed Ahmed, Zarlish Attique, Saad Ur Rehman, Ayesha Khan, Hussain Ali, Muhammad Rizwan, Anum Munir, Arshad Mehmood Khan, Faiza Siddique, Azhar Mehmood, Faisal Nouroz, Sajid Khan , J Biomol Struct Dyn2020 Jun 22;1-11.

doi: 10.1080/07391102.2020.1778534. Online ahead of print.

Abstract

An rare pandemic of viral pneumonia occurs in December 2019 in Wuhan, China, which is now recognized internationally as Corona Virus Disease 2019 (COVID-19), the etiological agent classified as Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2). According to the World Health Organization (WHO), it has so far expanded to more than 213 countries/territories worldwide. Our study aims to find the viral peptides of SARS-COV-2 by peptide mass fingerprinting (PMF) in order to predict its novel structure and find an inhibitor for each viral peptide. For this reason, we calculated the mass of amino acid sequences translated from the SARS-CoV2 whole genome and identify the peptides that may be a target for inhibition. Molecular peptide docking with Moringa oleifera, phytochemicals (aqueous and ethanolic) leaf extracts of flavonoids (3.56 ± 0.03), (3.83 ± 0.02), anthraquinone (11.68 ± 0.04), (10.86 ± 0.06) and hydroxychloroquine present therapy of COVID-19 in Pakistan for comparative study. Results indicate that 15 peptides of SARS-CoV2 have been identified from PMF, which is then used as a selective inhibitor. The maximum energy obtained from AutoDock Vina for hydroxychloroquine is -5.1 kcal/mol, kaempferol (flavonoid) is -6.2 kcal/mol, and for anthraquinone -6 kcal/mol. Visualization of docking complex, important effects are observed regarding the binding of peptides to drug compounds. In conclusion, it is proposed that these compounds are effective antiviral agents against COVID-19 and can be used in clinical trials.

Particulate Multivalent Presentation of the Receptor Binding Domain Induces Protective Immune Responses Against MERS-CoV

Nisreen M A Okba, Ivy Widjaja, Brenda van Dieren, Andrea Aebischer, Geert van Amerongen, Leon de Waal, Koert J Stittelaar, Debby Schipper, Byron Martina, Judith M A van den Brand, Martin Beer, Berend-Jan Bosch, Bart L Haagmans, Emerg Microbes Infect2020 Dec; 9(1):1080-1091.

doi: 10.1080/22221751.2020.1760735.

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is a WHO priority pathogen for which vaccines are urgently needed. Using an immune-focusing approach, we created self-assembling particles multivalently displaying critical regions of the MERS-CoV spike protein ─fusion peptide, heptad repeat 2, and receptor binding domain (RBD) ─ and tested their immunogenicity and protective capacity in rabbits. Using a “plug-and-display” SpyTag/SpyCatcher system, we coupled RBD to lumazine synthase (LS) particles producing multimeric RBD-presenting particles (RBD-LS). RBD-LS vaccination induced antibody responses of high magnitude and quality (avidity, MERS-CoV neutralizing capacity, and mucosal immunity) with cross-clade neutralization. The antibody responses were associated with blocking viral replication and upper and lower respiratory tract protection against MERS-CoV infection in rabbits. This arrayed multivalent presentation of the viral RBD using the antigen-SpyTag/LS-SpyCatcher is a promising MERS-CoV vaccine candidate and this platform may be applied for the rapid development of vaccines against other emerging viruses such as SARS-CoV-2.

Elucidation of Cellular Targets and Exploitation of the Receptor Binding Domain of SARS-CoV-2 for Vaccine and Monoclonal Antibody Synthesis

Abdul Mannan Baig, Areeba Khaleeq, Syeda Hira, J Med Virol2020 Jun 23.

doi: 10.1002/jmv.26212. Online ahead of print.

Abstract

The pandemic caused by novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) has resulted in over 452,822 deaths in the first twenty days of June 2020 due to the coronavirus virus disease 2019 (COVID-19). The SARS-CoV-2 uses the host angiotensin-converting enzyme 2 (ACE2) receptor to gain entry inside the human cells where it replicates by using the cell protein synthesis mechanisms. The knowledge of the tissue distribution of ACE2 in human organs is therefore important to predict the clinical course of the COVID-19. Also important is the understanding of the viral receptor-binding domain (RBD) a region within the spike (S) proteins that enable the entry of the virus into the host cells to synthesize vaccine and monoclonal antibodies (mAbs). We performed an exhaustive search of human protein databases to establish the tissues that express ACE2 and performed an in-depth analysis like sequence alignments and homology modeling of the spike protein (S) of the SARS-CoV-2 to identify antigenic regions in the RBD that can be exploited to synthesize vaccine and mAbs. Our results show that ACE2 is widely expressed in human organs that may explain the pulmonary, systemic, and neurological deficits seen in COVID-19 patients. We show that though the S protein of the SARS-CoV-2 is a homolog of S protein of SARS-CoV-1, it has regions of dissimilarities in the RBD and transmembrane segments. We show peptide sequences in the RBD of SARS-CoV-2 that can bind to the MHC alleles and serve as effective epitopes for vaccine and mAbs synthesis.